The central hypothesis of this proposal is that controlling the function of CD4+25+ reguhatory T cells (Treg) in vivo will abrogate cellular and humoral immune responses to human coagulation factor IX (hF.IX) antigen, which is administered by adeno-associated viral (rAAV) gene transfer. Because markers of Treg cells have only recently become available, it is now possible to enhance the number of activated Treg cells in vivo. The experiments proposed in this application are designed to examine the contributions and limitations of GITR and GITR-Ligand based on/off switches of Treg cell suppression of cell-mediated and humoral immune responses elicited by rAAV-hF.IX gene transfer. Secondly, adoptive transfer based cell therapy with Treg cells will be applied to control the rAAV-hF.IX induced cell-mediated responses. Third a strategy that targets activated cytopathic reactive T cells and spares immunoregulatory networks will be adapted from the transplantation field. The experiments proposed in this application are grouped in the following specific aims: Specific Aim #1: To test the hypothesis that administering a soluble GITR-Ligand-Fc fusion protein [Fc- GITR-L] inactivates Treg cell functions and thus enhances humoral and cellular immune responses to rAAV-hF. IX. Specific Aim #2: To test the hypothesis that treatment with a monoclonal antibody directed at mouse GITR-L enhances Treg cell suppression and immune tolerance to hF.IX antigens and AAV capsid antigen. Specific Aim #3: To test the hypothesis that suppression and/or deletion of potentially pathogenic T cells facilitates Treg mediated immunologic tolerance towards hF.IX.. Together these experiments should clarify the role of Treg cell controlled pathways in rAAV hF.IX gene therapy. The results of these studies should suggest therapeutic strategies that can immediately be applied to hemophilia patients.